Our overall goal in this project is to characterize the functional contributions of cyclin-dependent kinase 5 (Cdk5)-mediated neuronal phosphorylation to the nervous system. Neuronal phosphorylation plays a crucial role in neuronal development and function, such as sensory recognition, behavioral control, memory, and learning. Most importantly, recent evidence suggests that abnormal phosphorylation in neurons is involved in the pathogenic mechanisms underlying many neurological diseases and nociception. Our main focus has been directed toward elucidating the contribution of Cdk5 in pain signaling. Cdk5, a member of a large family of proline-directed serine/threonine protein kinases, was originally cloned by homology to other members of the Cdk family that are major regulators of cell-cycle progression. However, the involvement of Cdk5 in cell-cycle regulation has never been identified. Instead, it appears to play a major role in the nervous system. The neuronal specificity of Cdk5 kinase activity is achieved through the association with its activators, either p35 or p39, which are predominantly expressed in postmitotic neurons. To date, about 50 proteins with diverse functions have been identified as substrates of Cdk5, implicating its role in the regulation of a wide range of neuronal functions. Apart from the critical role of Cdk5 in normal brain functions, deregulation of Cdk5 is involved in the pathogenesis of neurodegenerative diseases such as Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Parkinson's disease, and drug addiction. We have used a variety of functional genomic techniques and molecular approaches, including conventional and conditional gene targeting, analysis of protein-protein interactions, as well as behavioral tests in mice, to address these questions. We believe our present studies will not only contribute to a greater understanding of the molecular basis of Cdk5 functions in the brain, but also will help future efforts toward the development of more effective diagnosis and treatments for debilitating neurodegenerative diseases, drug addiction, and chronic pain. [unreadable] [unreadable] Apart from its role in developing brain, Cdk5 has also been proposed as one of the possible molecules contributing to neuronal death. Disturbances in cellular calcium homeostasis induce the proteolytic cleavage of p35 to its truncated product, p25. This cleavage is achieved through the activation of the calcium-dependent cysteine protease, calpain, which eventually causes the mislocalization of Cdk5 in the cellular compartments and its prolonged activation. This deregulation of Cdk5 activity has been implicated in the pathogenesis of certain neurodegenerative diseases such as Alzheimer's and Parkinson's disease. Consistent with the hypothesis that Cdk5/p25 is an upstream regulator in the cascade leading to neuronal death in Alzheimer!|s disease, blocking Cdk5 activity prevented neuronal cell death induced by amyloid beta-peptide in vitro. Thus, Cdk5 has attracted much attention as a target for pharmacological intervention against neuronal cell death. Combining the potential utility of Cdk5 inhibitors for neuroprotective strategies with the possible diverse functions of Cdk5 in adult brain necessitates knowing the consequences of Cdk5 deficiency in postnatal brain as well as in developing brain. However, the perinatal lethality of Cdk5-/- mice has hampered the delineation of the precise functions of Cdk5 in postnatal brain. To circumvent this problem, we employed the cre-loxP recombination technique to generate a Cdk5 conditional knockout mouse in which Cdk5 was deleted in neurons of the forebrain, by expression of cre recombinase directed by the promoter of the CaMKII alpha gene encoding the alpha-subunit of the calcium/calmodulin-dependent kinase II (CaMKII). This strategy led to inactivation of Cdk5 specifically in the forebrain, including the olfactory bulb, cerebral cortex, hippocampus and striatum, as early as embryonic day (E)12.5. Cdk5 conditional mutant mice were viable, but exhibited complex neurological deficits such as dyskinesia of the limbs, tremors, seizures and hyperlocomotion. These neurological deficits were associated with neurodegenerative changes in the forebrain, which were accompanied by aberrant activation of glial cells including astroglia and microglia, leading to increased production of tumor necrosis factor-alpha, a pro-inflammatory cytokine. Furthermore, our search for the molecules responsible for the microglial activation identified Cdk5 as a negative regulator of tissue-type plasminogen activator, a serine protease known to be involved in microglial activation, seizure spreading and neuronal cell death. Our data indicate that Cdk5 deficiency leads to neurodegeneration in the postnatal brain, which may be attributed to some extent to tPA-mediated microglial activation.[unreadable] [unreadable] Pain is a combination of sensory (discriminative) and affective (emotional) components. The sensory component of pain is defined as nociception and is required for the survival and maintenance of the integrity of the organism. However, sustained or chronic pain, particularly in humans, can result in secondary symptoms such as anxiety, depression, and a marked decrease in the quality of life. Specific cell types and several molecules have been identified that detect and regulate nociceptive activity. Additionally, the parallel pathways that distribute nociceptive information to limbic or sensory areas of the forebrain have been elucidated, but the underlying molecular mechanisms remain unclear. So far, studies using genetically modified mice, antisense knockdowns in cells, gene expression assays (including DNA microarray-based expression profiling), and linkage mapping have identified several genes whose expression levels are directly or indirectly affected during pain sensation and/or are involved in modulating pain. As a result, the number of proteins encoded by these genes continues to expand and requires further investigation of their participation in pain pathways.[unreadable] [unreadable] Although Cdk5 regulates crucial neuronal functions, its deregulation, via the calpain-dependent cleavage of its activator p35, has been implicated in neurodegenerative disorders such as Alzheimer's and Parkinson's disease. Patients affected by these diseases not only exhibit impaired neuronal functions but also endure chronic pain. Until now, there has been no direct evidence indicating the involvement of Cdk5 in nociceptive processes. However, several known substrates and proteins interacting with Cdk5 have been linked to nociceptive pathways, suggesting that Cdk5 may be involved directly or indirectly in nociception. In pain signaling, much attention has been focused on the signal transduction mechanisms in primary afferent neurons responsible for the modulation of nociceptive transmission. We identified the expression of Cdk5 and its activator, p35, in nociceptive neurons, that is modulated during a peripheral inflammatory response. Increased calpain activity in sensory neurons following inflammation resulted in the cleavage of p35 to p25 and, consequently, elevation of Cdk5 kinase activity. The p35 knockout mice (p35-/-) which exhibit significantly decreased Cdk5 activity, showed delayed responses towards painful thermal stimulation compared to their wild-type controls. In contrast, mice overexpressing p35 (Tgp35) with elevated levels of Cdk5 activity were more sensitive to painful thermal stimuli than controls. In conclusion, our data demonstrate a role for Cdk5/p35 activity in primary afferent nociceptive signaling, suggesting that Cdk5/p35 may be a target for development of analgesic drugs.